You can use xs:uniqueto restrict Reference_numberas unique:
<?xml version="1.0" encoding="utf-8"?>
<xs:schema attributeFormDefault="unqualified"
elementFormDefault="qualified"
xmlns:xs="http://www.w3.org/2001/XMLSchema">
<xs:element name="Items">
<xs:complexType>
<xs:sequence>
<xs:element maxOccurs="unbounded" name="Item">
<xs:complexType>
<xs:sequence>
<xs:element name="Name" type="xs:string" />
<xs:element name="Reference_number" type="xs:string" />
<xs:element name="Price" type="xs:float" />
</xs:sequence>
</xs:complexType>
</xs:element>
</xs:sequence>
</xs:complexType>
<xs:unique name="UniqueItemRefNum">
<xs:selector xpath="Item" />
<xs:field xpath="Reference_number" />
</xs:unique>
</xs:element>
</xs:schema>
Then, for example, the following XML is valid:
<?xml version="1.0" encoding="utf-8"?>
<Items xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"
xsi:noNamespaceSchemaLocation="try.xsd">
<Item>
<Name>name1</Name>
<Reference_number>r1</Reference_number>
<Price>1.00</Price>
</Item>
<Item>
<Name>name2</Name>
<Reference_number>r2</Reference_number>
<Price>2.00</Price>
</Item>
</Items>
But the following XML will be invalid (note the duplicate Reference_number):
<?xml version="1.0" encoding="utf-8"?>
<Items xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance"
xsi:noNamespaceSchemaLocation="try.xsd">
<Item>
<Name>name1</Name>
<Reference_number>r1</Reference_number>
<Price>1.00</Price>
</Item>
<Item>
<Name>name2</Name>
<Reference_number>r1</Reference_number>
<Price>2.00</Price>
</Item>
</Items>
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